The E3 Ligase TRIM4 Facilitates SET Ubiquitin‐Mediated Degradation to Enhance ER‐α Action in Breast Cancer

Abstract Estrogen receptor alpha (ER‐α) action is critical for hormone‐dependent breast cancer, and ER‐α dysregulation can lead to the emergence of resistance to endocrine therapy. Here, it is found that TRIM4 is downregulated in tamoxifen (TAM)‐resistant breast cancer cells, while the loss of TRIM4 is associated with an unfavorable prognosis. In vitro and in vivo experiments confirm that TRIM4 increased ER‐α expression and the sensitivity of breast cancer cells to TAM. Mechanistically, TRIM4 is found to target SET, and TRIM4‐SET interactions are mediated by the RING and B‐box domains of TRIM4 and the carboxyl terminus of SET. Moreover, it is determined that TRIM4 catalyzed the K48‐linked polyubiquitination of SET (K150 and K172), promoting its proteasomal degradation and disassociation from p53 and PP2A. Once released, p53 and PP2A are able to further promote ESR1 gene transcription and enhance mRNA stability. Moreover, univariate and multivariate Cox proportional hazards regression analyses confirm that TRIM4 expression is an independent predictor of overall survival and recurrence‐free survival outcomes in patients with ER‐α positive breast cancer. Taken together, the data highlights a previously undiscovered mechanism and suggest that TRIM4 is a valuable biomarker that can be analyzed to predict response to endocrine therapy in breast cancer patients.


Supplementary Text
Materials and methods qPCR Trizol (Invitrogen) was used to extract total RNA from cells based on provided directions, after which reverse transcriptase (Takara, Shiga, Japan) was used to prepare cDNA, and qPCR analyses were performed with a SYBR Green RT-PCR kit (Roche, Basel, Switzerland). GAPDH expression was used to normalize gene expression. All qPCR primers used in this study are listed in Supplementary Table 1.

Mass spectrometry analyses.
For mass spectrometry analysis, IP was performed with the whole cell lysates in MCF7 cells transfected with Flag-tagged TRIM4 or control plasmids. The IP proteins were resolved by SDS-PAGE, followed by Coomassie staining and the next mass spectrometry analyses were performed by Jingjie PTM BioLab (Hangzhou, China).

Immunofluorescent (IF) and confocal microscopy
IF analyses were performed as in a prior report [1]. Briefly, MCF7 cells were added onto glass coverslips in 24-well plates. Following TRIM4-GFP and SET-Myc plasmid transfection, cells were incubated for 90 min with anti-Myc without permeabilization. They were then washed two times with PBS, probed with a secondary Alexa Fluor 633-conjugated antibody for 30 min and imaged with a laser-scanning confocal microscope (Nikon confocal microscopy, Eclipse Ti A1).

Colony formation assay.
Colony forming ability were assessed using a colony formation assay which was performed as previously described [2]. The transfected cells were seeded into 6 cm plates at a density of 5×10 3 cells/well for 24 h, followed by treatment with indicated TAM for 48 h. After cultured with new medium for 2 weeks, the cells were then washed twice with cold PBS, fixed with methanol-glacial acetic acid stationary solution (3:1), and stained with 0.5% crystal violet solution. Pictures were imaged and counted.

Transwell.
Transwell assay was carried out as previously described [3]. Briefly, after transfection, cells underwent a 6 h serum starvation step, 1×10 5 MCF7/TR or T47D/TR cells and 2×10 5 MCF-7 or T47D cells were suspended in 200μL serum-free medium and seeded into the inside of each transwell insert (Corning Costar, USA), while 700 μL medium containing 20% FBS was placed in the lower well. After incubation for 24-72 h, the infiltrating cells on the lower surface were fixed with methanol and stained with 0.1% crystal violet. Cell invasion assay was conducted using the same procedure as in the cell migration assay, except that the inside of each insert was coated with Matrigel (BD Biosciences, San Jose, CA, USA).
Puromycin (0.5 mg/mL; Sigma) was then used to select cells for 3 weeks, after which the organoids were imaged and counted via microscopy (20×; Zeiss ObserverZ1). Organoids were then treated for an additional 4 days with TAM (15 µM) and imaged at 20×magnification.

Molecular modelling and protein-protein docking.
The amino-acid sequences of TRIM4 (NP_148977.2) and SET (NP_001116293.1) retrieved from the NCBI database were used as targets for homology modeling using I-TASSER (H) Correlations between TRIM4 expression and ER-α status in breast cancer cohorts (ER-α positive: n=398, ER-α negative: n=117). Data information: data were presented as mean ± SD.